The disclosed invention is generally in the field of nucleic acid amplification.
DNA molecular cloning is routinely carried out using plasmid, phage, or viral vectors that replicate inside cells. Genomic cloning is routinely carried out using vectors that replicate inside cells. While existing cloning methods work quite well for most genomic fragments, certain DNA domains tend to suffer alterations, notably deletions or rearrangements. A method, in which individual DNA molecules are cloned in solution by serial dilution and subsequent PCR amplification from tubes containing single molecules has been described (Lukyanov et al., Nucleic Acid Research 24:2194-2195 (1996)). A method has also been described for cloning RNA populations derived from single RNA molecules in an immobilized medium (Chetverina and Chetverin, Nucleic Acids Research 21:2349-2353 (1993)). While both of these methods allow in vitro cloning, neither is practical for cloning of large fragments.
A number of methods have been developed for exponential amplification of nucleic acids. These include the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and amplification with Q.beta. replicase (Birkenmeyer and Mushahwar, J. Virological Methods, 35:117-126 (1991); Landegren, Trends Genetics 9:199-202 (1993)).
Current methods of PCR amplification involve the use of two primers which hybridize to the regions flanking a nucleic acid sequence of interest such that DNA replication initiated at the primers will replicate the nucleic acid sequence of interest. By separating the replicated strands from the template strand with a denaturation step, another round of replication using the same primers can lead to geometric amplification of the nucleic acid sequence of interest. PCR amplification has the disadvantage that the amplification reaction cannot proceed continuously and must be carried out by subjecting the nucleic acid sample to multiple cycles in a series of reaction conditions. PCR also has the disadvantage that the length of nucleic acid that can be effectively amplified is limited.
Accordingly, there is a need for a cloning and amplification method that allows amplification of longer nucleic acid segments and that is less complicated, are more reliable, and produces greater amplification in a shorter time.
It is therefore an object of the disclosed invention to provide an in vitro method of cloning and amplifying a target nucleic acid sequence in a continuous, isothermal reaction.
It is another object of the disclosed invention to provide an in vitro method of cloning and amplifying a target nucleic acid sequence where multiple copies of the target nucleic acid sequence are produced in a single amplification cycle.
It is another object of the disclosed invention to provide a kit for cloning and amplifying a target nucleic acid sequence in a continuous, isothermal reaction.